CD9+ Regulatory B Cells Induce T Cell Apoptosis via IL-10 and Are Reduced in Severe Asthmatic Patients

Abstract

CD9 was recently identified as a marker of murine IL-10-competent regulatory B cells. Functional impairments or defects in CD9⁺ IL-10-secreting regulatory B cells are associated with enhanced asthma-like inflammation and airway hyperresponsiveness. In mouse models, all asthma-related features can be abrogated by CD9⁺ B cell adoptive transfer. We aimed herein to decipher the profiles, features, and molecular mechanisms of the regulatory properties of CD9⁺ B cells in human and mouse. The profile of CD9⁺ B cells was analyzed using blood from severe asthmatic patients and normal and asthmatic mice by flow cytometry. The regulatory effects of mouse CD9⁺ B cells on effector T cell death, cell cycle arrest, apoptosis, and mitochondrial depolarization were determined using yellow dye, propidium iodide, Annexin V, and JC-1 staining. MAPK phosphorylation was analyzed by western blotting. Patients with severe asthma and asthmatic mice both harbored less CD19⁺CD9⁺ B cells, although these cells displayed no defect in their capacity to induce T cell apoptosis. Molecular mechanisms of regulation of CD9⁺ B cells characterized in mouse showed that they induced effector T cell cycle arrest in sub G0/G1, leading to apoptosis in an IL-10-dependent manner. This process occurred through MAPK phosphorylation and activation of both the intrinsic and extrinsic pathways. This study characterizes the molecular mechanisms underlying the regulation of CD9⁺ B cells to induce effector T cell apoptosis in mice and humans via IL-10 secretion. Defects in CD9⁺ B cells in blood from patients with severe asthma reveal new insights into the lack of regulation of inflammation in these patients.

Keywords: CD9+ B cells; apoptosis; effector T cells; regulatory B cells; severe asthma.

Figure 1

B lymphocyte subpopulations in the blood of severe asthmatic patients. (A) Gating strategy used after immunostaining to determine all B cell subsets. (B) Assessment of CD19⁺ B lymphocytes, CD19⁺CD27⁺ memory cells, CD19⁺CD27⁻ naive cells, CD19⁺CD24⁻CD38⁺ plasma cells, CD19⁺CD24^hiCD38^hi transitional cells, and CD9⁺ B cells in 10 healthy volunteers (HV) and 9 severe asthmatic patients (SA) (*p < 0.05, **p < 0.01). (C) Expression of the mean fluorescence intensity of CD9 in transitional and non-transitional B cell subsets (***p < 0.001).

Figure 2

Percentage and regulatory properties of CD9⁺ B cells in asthmatic mice. (A) Induction protocol in asthma mice: House dust mite model. (B) Percentage of CD9⁺ B cells among CD19⁺ cells in the spleen and lung of control and asthmatic mice (n = 4, *p < 0.05). (C) Gating strategy used to remove B cells from the analysis by CD4 FITC staining. (D) After 48 h of activation, splenic CD3⁺CD4⁺CD25⁻ effector T cells from asthmatic and naive Balb-c mice were co-cultured for 48 h with CD19⁺CD9⁺ or CD19⁺CD9⁻ B cells or alone as controls. Cells were stained with yellow dye to measure T cell death induced by CD9⁺ or CD9⁻ B cells. Percentage of Annexin V-positive T cell staining (n = 6, *p < 0.05). (E) Percentage of T cell death induction by CD19⁺CD9⁺ or CD19⁺CD9⁻ B cells (ns, non-significant).

Figure 3

Effects of CD9^+/− B cells on T cell proliferation and death. After 48 h of activation, splenic CD3⁺CD4⁺CD25⁻ effector T cells from Balb-c mice were co-cultured for 48 h with CD19⁺CD9⁺ or CD19⁺CD9⁻ B cells at a 1:1 ratio or alone as controls. Cells were stained with CD4 FITC antibody to remove B cells from the analysis. (A) Cells were stained with propidium iodide to measure T cell cycle stages. Representative staining of the different cell cycle stages. (B) Percentage of T cells in the different cell cycle stages (n = 6).

Figure 4

Effects of CD9^+/− B cells on T cell apoptosis. (A–C) After 48 h of activation, splenic CD3⁺CD4⁺CD25⁻ effector T cells from Balb-c mice were co-cultured for 48 h with CD19⁺CD9⁺ or CD19⁺CD9⁻ B cells or alone as controls. During co-culture, the cells were also treated with 2 μg/μL KMC8 anti-CD9 antibody, 50 nM Z-VAD, 10 ng/mL IL-10, 10 μg/mL anti–IL-10, 10 μg/mL anti–TGF-β1, or 20 μg/mL anti-Fas ligand. Cells were also co-cultured in trans-wells. The cells were stained with CD4 antibody to remove B cells from the analysis and with Annexin V to measure T cell apoptosis. (A) Representative results of Annexin V staining. (B,C) Percentage of Annexin V-positive T cells in each co-culture condition (n = 6, *p < 0.05, **p < 0.01). (D) After 48 h of activation, CD3⁺CD4⁺CD25⁻ effector T cells from 9 healthy volunteers were co-cultured for 48 h with CD19⁺CD9⁺ or CD19⁺CD9⁻ B cells or alone as controls. Cells were also treated with 20 μg/mL anti–IL-10 and stained with CD4 antibody to remove B cells from the analysis and stained with Annexin V to measure T cell apoptosis. Percentage of Annexin V-positive T cells in the different co-culture conditions (n = 9; *p < 0.05).

Figure 5

Effects of CD9⁺ B cells on MAPK phosphorylation, Bid and caspase cleavage and mitochondrial depolarization. After 48 h of activation, splenic CD3⁺CD4⁺CD25⁻ effector T cells from Balb-c mice were co-cultured for 48 h with CD19⁺CD9⁺, CD19⁺CD9⁻ B cells or alone as controls. (A) B cells were removed from the analysis by CD4 staining. Phosphorylation of MAPK p38, JNK, and ERK1/2 in CD3⁺CD4⁺CD25⁻ effector T cells was assessed by flow cytometry. Representative results of MAPK staining. (B) CD3⁺CD4⁺CD25⁻ effector T cells were negatively selected using MACS columns. Cleavage of caspase 8, 9, and 12 was assessed by western blotting, and the ratios of cleaved/pro-caspases were calculated. (C) CD3⁺CD4⁺CD25⁻ effector T cells were negatively selected by MACS columns. Bid cleavage was assessed by western blotting. Actin was used as the loading control. (D) Mitochondrial depolarization was assessed by JC-1 staining. Representative results of JC-1 staining and percentage of monomeric JC-1 cells in all culture conditions (n = 5, *p < 0.05).